The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

PURPOSE: Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. METHODS: Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of < or =270 microm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09%). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. CONCLUSION: Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.

Induction of genotoxic effects and modulation of the intracellular calcium level in syrian hamster embryo (SHE) fibroblasts caused by ochratoxin A.

The mycotoxin ochratoxin A (OTA) is a naturally occuring contaminant of food. The genotoxic status of OTA is still controversial because contradictory results were obtained in various microbial and mammalian gene mutation assays. In this study, OTA was investigated to examine its potency to induce micronuclei (MN) in SHE cells. The SHE-micronucleus assay revealed that OTA induces MN in a dose- and time-dependent manner. The results of kinetochore analysis revealed that mainly clastogenic events are involved in OTA genotoxicity. Induction of mitotic disturbances can be closely related to changes of the intracellular calcium concentration ([Ca2+]i). The investigated time course of OTA-induced [Ca2+]i changes revealed that the obtained signal is a short spike signal resembling physiological responses. In the absence of extracellular calcium, a long-lasting signal indicates possible damage to intracellular calcium stores or channels. Our data show that the OTA-induced [Ca2+]i rise is caused by Ca2+ -release from intracellular stores as well as Ca2+ influx from extracellular area. Finally, the influence of the changed intracellular calcium level on the actin cytoskeleton was investigated. Visualization of the actin filaments revealed time- and concentration-dependent effects. Cell shrinkage and depolymerized filaments were observed. We conclude that OTA disrupts actin filaments by a direct irreversible binding to actin.

Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.

OBJECTIVES: The investigation centers on whether there is a reperfusion-induced specific cardiac inflammatory reaction after bypass surgery. BACKGROUND: Cardiopulmonary bypass (CPB) leads to systemic inflammation. Additionally, cardiac inflammation due to reperfusion could occur. Knowledge about nature and time course of this reaction might help to develop cardioprotective interventions. METHODS: In 12 patients receiving coronary bypass grafts, arterial and coronary venous blood was obtained before onset of CPB, and 1, 5, 10, 25, 35 and 75 min after cardiac reperfusion. Plasma levels of IL6 and IL8 were measured by immunoassay. CD11b, CD41, and CD62 on blood cells were quantified by flow cytometry. Measurement of CD41, a platelet marker, on neutrophils and monocytes allowed detection of leukocyte-platelet microaggregates. RESULTS: Transcardiac veno-arterial difference of IL6 rose in the 10th and 25th min of reperfusion (from 0 to 7 pg/ml; p < 0.05), and after 75 min (15 pg/ml). IL8 did not change. CD11b on neutrophils (PMN) decreased transcardially to 95, 88 and 82% of the initial level in the 5th, 10th, and 75th min, respectively, suggesting sequestration of activated neutrophils. CD62 on platelets rose about 30% in the 75th min. Initially, leukocyte-platelet microaggregates were formed during coronary passage (+31% of the arterial level for PMN, +23% for monocytes). During reperfusion, coaggregates were retained (PMN: -1% and -7% in the 5th and 10th min, monocytes: -22%, -13% and -12% in the 1st, 5th and 10th min. CONCLUSIONS: During early reperfusion after aortic declamping, the coronary bed is already a source of proinflammatory stimuli and target for activated leukocytes, partly in conjunction with platelets. Mitigation of these phenomena might help to improve cardiac function after CPB especially in patients at risk.

Modulation of the intracellular calcium level in mammalian cells caused by 17beta-estradiol, different phytoestrogens and the anti-estrogen ICI 182780.

In previous investigations it was shown that the synthetic estrogen diethylstilbestrol (DES) induces a rise of the intracellular calcium level ([Ca2+]i) in C6 rat glioma cells [P. Tas, H. Stopper, K. Koschel, D. Schiffmann, Influence of the carcinogenic oestrogen diethylstilboestrol on the intracellular calcium level in C6 rat glioma cells. Toxic. In vitro 5 (1991) 463-465] which is accompanied by changes of the arrangement of the cytoskeleton. In the present study, we compared the induction of these effects in COS (monkey kidney cells) lacking estrogen receptors (ER) with those in RUCA-I (rat endometrial carcinoma) cells containing ER. The [Ca2+]i in RUCA-I and COS cells following 17beta-estradiol (ES), genistein (GEN), daidzein (DZ) and coumestrol (CES) treatment was analyzed. A significant increase of [Ca2+]i induced by all compounds was observed in RUCA-I cells. No effects were detected in COS cells after ES and GEN treatment. The anti-estrogen ICI 182780 completely blocked the ES-and GEN-induced rise of [Ca2+]i. Dose and time dependencies of changes of calcium levels were analyzed and a biphasic response could be observed. The actin staining showed disintegrated stress fibers in RUCA-I cells. The degree of the observed effects correlates with the known estrogenicity of the applied compounds (DES > ES > GEN). It remains to be elucidated whether or not the effects observed are mediated by the "classic" genomic estrogen receptor pathway or by alternate nongenomic or receptor-independent pathways.

Technical refinements in myocardial protection: infants--adults.

The aim of intraoperative protection is to prevent damage to function and structure of the myocardium. None of the methods employed today can guarantee this, on the other hand the result of any surgical intervention has to be regarded as a multifactorial process, myocardial function in particular depending on e.g. the preoperative state, the mode of protection, temperature of the patient, collateral flow, unloading of the left ventricle, and other factors during ischemic arrest. Daily use of cardioplegic solutions requires standardized procedures keeping it safe and simple. Thus we use in adults 1000ml of 2 degrees Bretschneider solution infusing it at rate of 80 - 120ml/min 8-10 minutes; in infants and children 40ml/kg as a single infusion are administered. The temperatures of the patients are various. As a result of myocardial protection, in the follow-up, besides survival, myocardial function should be a decisive parameter. Of particular interest are the results in patients with preoperatively reduced myocardial function and the effect of myocardial protection techniques. In addition, examples of long-term survivors after congenital operations will be discussed in accordance to the cardioplegic regimen used. Recent work has shown that reperfusion may aggravate the damage imposed on the heart during ischemia. An additional inflammatory reaction is observed which may compromise function. There is evidence that, under experimental and clinical conditions, the donation of nitric oxide may limit the amount of postischemic cardiac inflammation. Simple, safe, and reproducible myocardial protection together with careful, sophisticated, and perfect operative technique are the main requirements for successful cardiac surgery.

[Keratinocyte density of the cornea in vivo. Automated measurement with a modified confocal microscopy MICROPHTHAL]. / Keratozytendichte der In-vivo-Kornea. Automatische Messung mit einem modifizierten konfokalen Mikroskop MICROPHTHAL.

BACKGROUND: The MICROPHTHAL is a confocal slit light scanning microscope for a non-invasive in-vivo examination of corneal structures of human eyes. With this instrument even thin layers of corneal tissue can be imaged in good quality. Otherwise, blurring of single frames and deviations from the z-axis in video-sequences caused by high speed movements of the eye would normally prevent a measurement the density of keratocytes in the cornea. The goal of the investigation was optical pachymetry, the automatical measurement of the keratocytes density and a 3D-dimensional reconstruction of the central cornea in-vivo under constant imaging conditions. MATERIALS AND METHODS: We developed a low-vacuum suction cup system for stabilizing the eye in front of the microscope objective during the z-scan through the cornea. A stepmotor shifting system for the objective locates inside the suction cup with a central hole was installed underneath them icroscope. Control of this system via computer facilitated shifting the focal plane along the z-axis. The layer images were recorded using a S-VHS-tape and saved on the PC. The digital analysis was performed using a special software to automatically and off-line evaluate the density of keratocytes in combination with the 3D-reconstruction. The software also corrected the background illumination and small axial jitter. After this procedure the keratocytes density and the 3D-reconstruction in 70 images of the z-scan were calculated. We examined 47 corneas of 25 healthy probands. The range of age was 25-56 years. Independent control evaluation of the video sequences were taken manually on an INDIGO HIGH IMPACT workstation. RESULTS: By assign all keratocytes to the corneal measurement volume we found a averaged density of 15,730 cells/mm3 in the central cornea. The averaged thickness of the cornea was 0.556 mm. The control valuation of identical video-sequences on the workstation accomplished the same result of 16,000 keratocytes/mm3, also similar the result of the automatically measurement with the modified software. CONCLUSIONS: This modification of the microscope is a promising in-vivo tool for optical pachymetry and quantitative examination of corneal microstructures. The stabilization effect of the low-vacuum suction cup system in the front of the microscope for computer-controlled valuation of the density profile of keratocytes and the 3D-reconstruction of a central corneal volume element has produced encouraging results. Characterization of pathophysiological changes in the distribution of keratocytes after excimer laser ablation for phototherapeutic or photorefractive keratectomy, for example, can be estimated without pain for the patients.

Morphometric measurements to quantify the cerulein induced hyperstimulatory pancreatitis of rats under the protective effect of lectins.

In preceding papers we demonstrated an inhibitory effect of wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) on the cholecystokinin (CCK) binding to the CCK receptor of rat pancreatic cells and also on the CCK induced Ca2+ release and alpha-amylase secretion in vitro as well as on pancreatic secretion of intact rats in vivo. In the present study we show the same inhibitory effect of both lectins on the cerulein pancreatitis of rats. This acute pancreatitis was induced by supramaximal injections (5 microg/kg/h i.v. or 10 microg/kg/h i.p.) of the CCK analogue cerulein in rats every hour. To monitor the degree of pancreatitis, we measured the number and diameter of injury vacuoles in the pancreatic acinar cells as one of the most important signs of this type of pancreatitis by light microscopic morphometry with two different systems on paraffin sections. Furthermore, the serum alpha-amylase activity was measured biochemically. We found a correlation between the diameter of vacuoles inside the acinar cells and the serum enzyme activity up to 24 h. The simultaneous i.p. administration of cerulein and WGA or UEA in a dosage of 125 microg/kg/h for 8 h led to a reduction of vacuolar diameter from 13.1+/-2.0 microm (cerulein) to 7.5+/-1.1 microm (cerulein + WGA) or 7.2+/-1.3 microm (cerulein + UEA). The serum amylase activity was reduced from 63.7+/-15.8 mmol/l x min (cerulein) to 37.7+/-11.8 (cerulein + WGA) or 39.4; +52.9; -31.1 (cerulein + UEA-I). Both parameters allow the grading this special type of pancreatitis to demonstrate the protective effect of the lectins.

Fluorescence microscopy and three-dimensional imaging of the porcine corneal keratocyte network.

BACKGROUND: Little is known about the spatial arrangement and the corresponding morphometric data describing the living keratocyte network. For determination of alterations in corneal diseases it is crucial to know the morphology of the keratocyte network in the healthy state. Porcine cornea was used as a model tissue because it allows the study of species differences. METHODS: Corneas from freshly enucleated pig eyes were stained with calcein AM and ethidium homodimer and examined by confocal laser scanning microscopy. High-resolution fluorescence images were used for three-dimensional reconstructions from which cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished and visualized in their spatial arrangement. Significant differences with respect to both shape and fluorescence intensity distribution of the cell bodies were found. Cell volume density was 7.7% in the anterior stroma, 13.7% in the central stroma and 11.8% in the posterior stroma. CONCLUSION: The technique described allowed good visualization of the spatial arrangement of the keratocyte network. Combined with morphometric methods, the analysis of the state of the cornea yields a quantitative description. The method is expected to be useful for the determination of morphological alterations in corneal disease or following surgical treatment.

Neuronal control of MHC class II inducibility in rat astrocytes and microglia.

We analysed the inducibility of major histocompatibility complex (MHC) class II molecules of astrocytes and microglia in organotypic hippocampus slice cultures of Lewis rats. Treatment with interferon-gamma (IFN-gamma) resulted in the induction of MHC class II molecules on microglia preferentially in the injured marginal zones of the slice culture, but only sporadically in areas containing intact neuronal architecture. In astrocytes, inducibility of MHC class II molecules was even more strictly controlled. IFN-gamma treatment induced MHC class II expression only in the slice culture zones containing degenerated neurons, and not in the presence of functional neurons. After suppression of spontaneous neuronal activity of the slice culture by the sodium channel blocker tetrodotoxin, MHC class II molecules on astrocytes could be induced by IFN-gamma in areas with intact neuronal architecture, and microglia cells exhibited a higher level of expression. These data suggest that loss of neurons could result in MHC class II inducibility of glial cells, and thus in increased immune reactivity of nervous tissue.

Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus.

The purpose of this study was the determination of morphological changes in the corneal epithelium and the keratocyte network in keratoconus. In all, 33 eyes of 19 patients were examined in vivo using the confocal slit-scanning microscope Microphthal. After penetrating keratoplasty, recipients' trephanates were stained with the Live/Dead kit and examined using the confocal laser-scanning fluorescence microscope Diaphot 300/Odyssey. The fluorescence images were reconstructed three-dimensionally. All findings were compared with data from healthy corneas. Morphological alterations were found only in the area of the corneal apex; obviously elongated superficial epithelial cells arranged in a whorl-like fashion were found. Near Bowman's membrane, highly reflective changes and fold-like structures were visible. The anterior stroma also showed an increased reflectivity. In the posterior stroma, typical findings were Vogt's striae and keratocytes with extremely long processes arranged nearly in parallel. In scarred stroma the keratocytes were spindle-shaped and arranged irregularly. The spatial organization of the living keratocyte network could be demonstrated through three-dimensional reconstructions.

Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response.

Mechanical loading of cells is of fundamental relevance in physiological processes and induces several functional responses in cells. Integrins, a family of adhesion receptors, which are responsible for the interaction with the extracellular matrix, may play a role in transmission of mechanical signals into cells. The osteogenic cell line U-2 OS expresses different integrin subunits which are uniformly distributed over the cell surface. We applied defined physical forces on individual integrin receptor subunits using paramagnetic microbeads coated with anti-integrin antibodies. Application of an inhomogeneous magnetic field consequently leads to a mechanical stress on the receptor. Intracellular Ca2+ increased when the alpha 2 or the beta 1 integrin subunits were stressed, whereas mechanical loading of the transferrin receptor had a significantly lower effect. This result indicates that forces specifically exerted to individual integrin receptors induce signal transduction pathways.

Operative correction of traumatic tricuspid insufficiency.

Between 1975 and 1991, 5 patients were operated for traumatic tricuspid insufficiency. The patients, all male, with the age at surgery of 15 to 61 years (mean 39 years), suffered previous nonpenetrating trauma to the chest 0.1 to 23 years (mean 13.2 years) earlier. Symptoms were known for 0 to 18 years (mean 10.5 years). The patients were in NYHA class II to IV (mean 2.9). Preoperative angiography showed moderate to severe tricuspid insufficiency. Mean right atrial pressure was 9.6 +/- 1.7 mm Hg and mean pulmonary artery pressure 14.0 +/- 2.4 mm Hg, the mean cardiothoracic ratio was 0.59 +/- 0.04, 4 patients were in sinus rhythm and 1 patient was in atrial fibrillation. Intraoperatively 4 patients showed rupture of the anterior chordae tendineae, 1 patient had multiple ruptures of the leaflets and the anterior papillary muscle, all tricuspid valves showed massive annular dilatation. A primary valvular reconstruction was undertaken in 3 patients of whom only 1 patient was successful in controlling tricuspid insufficiency. Finally, 4 tricuspid valves had to be replaced, 3 with a bioprosthetic and 1 with a mechanical valve. One patient died early, 2 patients died late during a total follow-up of 35.3 years after 7 and 9 years, 2 patients are alive 9 and 10 years after the operation and are presently in New York Heart Association class II to III and I. Traumatic tricuspid insufficiency is a rare event, but is not infrequently overlooked for a long period of time inspite of present symptoms. Results after operative correction seem to be comparable with the results in patients following tricuspid repair or replacement for functional and organic lesions.

Mineral fibers induce apoptosis in Syrian hamster embryo fibroblasts.

It is known that asbestos and other mineral fibers induce lung cancer and mesothelioma. However, the primary mechanisms of fiber-induced carcinogenesis still remain to be elucidated. Previous studies, including our own, have shown that asbestos causes specific mitotic disturbances, micronucleus formation and typical changes in chromatin structure resembling those of apoptosis. This effect has been considered as programmed cell death removing damaged or pre-cancerous cells. We investigated the induction of apoptosis by asbestos (amosite, crocidolite, chrysotile) and ceramic fibers. The typical ladder pattern of DNA fragments was identified by means of gel electrophoresis, the intracellular calcium concentration was measured and flow cytometry analyses were carried out to determine the percentage of apoptotic cells. The different fibers showed different potencies for the induction of apoptosis in Syrian hamster embryo (SHE) cells. Depending on the type of fiber applied 3-33% of cells underwent apoptosis. Chrysotile proved to be the most potent inducer of apoptosis compared to the other fibers. In addition, an increase intracellular calcium level was observed in apoptotic SHE cells. Chrysotile induced apoptosis after a considerably longer exposure time (66-72 h) than cisplatin (24 h). In view of these findings we hypothesize that chrysotile induces apoptosis resulting from long-term changes in intracellular regulation pathways.

Refolding of human class II major histocompatibility complex molecules isolated from Escherichia coli. Assembly of peptide-free heterodimers and increased refolding-yield in the presence of antigenic peptide.

The alpha- and beta-chains of the heterodimeric major histocompatibility complex molecules HLA-DRB5*0101 and DRB1*0101 were expressed separately in Escherichia coli. The cytoplasmic and membrane-spanning domains of both chains were replaced by oligohistidine tags to allow purification by metal chelate chromatography. The recombinant proteins were refolded to peptide-free, water-soluble heterodimers by removal of major amounts of detergents and concomitant reoxidiation of disulfide bonds. Correct conformation was documented by three criteria: (a) affinity binding experiments using the antibody L243, which is known to recognize a conformational epitope formed only by correctly associated heterodimers; (b) specific binding of peptides to the refolded molecules; (c) recognition of peptides bound to refolded HLA-DR molecules by T-cells as reflected by Ca2+ influx into T-cells and production of interferon-gamma. The refolding reaction did not absolutely depend on the presence of peptides. The yield of peptide-free heterodimers was 3.0%. However, the yield of refolded heterodimer was increased to 10% if refolding was performed in the presence of antigenic peptides.

BDNF and NT-3 induce intracellular Ca2+ elevation in hippocampal neurones.

The effects of neurotrophins on intracellular Ca2+ levels in rat hippocampal neurones were studied in vitro using fura-2 fluorescence microscopy. BDNF and NT-3, but not NGF, rapidly increased cytoplasmic Ca2+ concentrations in these neurones ten-fold to reach 1 microM. Moreover in some of the neurones both BDNF and NT-3 elicited Ca2+ responses, indicative of the presence of functional receptors for these neurotrophins in the same cell. In these cultures approximately 80% of the hippocampal neurones were stained with antibodies against full-length TrkB. The expression of functional TrkB was also confirmed by RNA analysis. These results demonstrate the presence of functional receptors for BDNF and NT-3 in hippocampal neurones.

High-dose aprotinin in cardiac surgery: three years' experience in 1,784 patients.

The effect of the proteinase-inhibitor aprotinin on blood loss and homologous blood requirement in cardiac surgery was investigated. In a prospective study, 902 adult patients were treated with high-dose aprotinin (total greater than 5 x 10(6) kallikrein inactivator units [KIU]; group A), while 882 patients without aprotinin administration served as the controls (group C). Both groups were operated on between January 1987 and October 1989, and included patients with primary coronary artery bypass grafting (n = 525 group C, n = 560 group A), valve replacement (n = 292 group C, n = 264 group A), or combined procedures (n = 65 group C, n = 78 group A), as well as cardiac reoperations (n = 91 group C, n = 110 group A). The average blood loss 36 hours postoperatively in the aprotinin group was 679 +/- 419 mL, compared with 1,038 +/- 671 mL in the control group (P less than 0.05). Total homologous blood requirement was also significantly less in group A (942 +/- 1,630 mL) compared with group C (1,999 +/- 2,283 mL) (P less than 0.05), a reduction of 53%. Serum creatinine concentrations did not show intergroup differences on the first postoperative day (group A, 1.2 +/- 0.7; group C, 1.3 +/- 0.5 mg/dL) or on discharge from the intensive care unit (ICU). Thus, impairment of renal function as a consequence of aprotinin treatment was not observed. Three patients developed signs of mild circulatory depression after injection of aprotinin, which responded promptly to vasopressor therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Reconstruction of atrioventricular valves in partial and total atrioventricular canal]. / Rekonstruktion der AV-Klappen beim partiellen und totalen AV-Kanal.

Among the anomalies of endocardial cushion defects, the partial (PCAVC) and common (CCAVC) AV canal present a special challenge to the cardiac surgeon. In particular, reconstruction of the AV valve can be difficult in CCAVC because of morphologic variations. Within 17 years, 383 patients with this disease were operated on. Early mortality of PCAVC is below 1%, and of CCAVC below 5%. A total of 90% of the survivors are in NYHA class I. In general, plastic reconstruction of the mitral valve is performed. The risk of reoperation for recurrent mitral incompetence in both groups is between 7% and 10%. Mitral valve replacement is a rare event (1.5%).

p21ras Oncogene Protein Selectively Increases Low-voltage-activated Ca2+ Current Density in Embryonic Chick Dorsal Root Ganglion Neurons.

p21ras protein resembles the alpha subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca2+ channels in vertebrate sensory neurons by p21ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca2+ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca2+ channels in chick dorsal root ganglion neurons, p21ras did not significantly affect HVA Ca2+ currents. To study the time course of p21ras action, guanosine triphosphate-preloaded p21ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 - 30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p21ras, which was found to be inactive in biological assays, enhanced LVA Ca2+ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca2+ channel by p21ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca2+ currents. The regulatory role of p21ras on LVA channels may therefore be particularly important during differentiation.

[Clinical experiences and long-term results after surgical treatment of type A thoracic aortic aneurysm]. / Klinische Erfahrungen und Langzeitergebnisse nach operativer Behandlung des thorakalen Aortenaneurysmas Typ A.

For aortic aneurysms type A operative treatment is the method of choice. During the past 15 years 128 patients with this diagnosis have been operated on. Early mortality was 22%, 77% of all dissections were emergencies. Main causes of death were multiorgan failure. Up to 117 months 8 patients died late in part due to additional ruptures. 17% of the survivors required reoperation up to 154 months. The probability of survival after 10 years is 0.78, in the group with dissections only 0.4. Therefore close follow-up of this patient group is necessary in order to recognize progression of the disease and induce elective operative treatment.